Cytotoxicity assays as tools to assess water quality in the Sinos River basin

Abstract

Cytotoxicity assays using cell cultures may be an alternative to assess biological toxicity of surface waters and may help to improve the control of water quality. This study compared two methods to prepare culture media for the exposure of Hep-2 cells to water samples collected from the Rolante River, an important affluent of the Sinos River. The toxicity was evaluated using the MTT and neutral red assays. Two methods were used to prepare culture media. In method 1, the sample was diluted at 1:1, 1:10, 1:100, 1:1000, 1:10.000 (v/v, sample/medium) in a standard culture medium; in method 2, water samples were used as the solvent for the culture medium, which was prepared at concentrations of 100, 80, 60, 40 and 20%. Semi-confluent cultures were then exposed to the media test for 24 hours, and cytotoxicity was determined immediately using the MTT and NR assays. Mitochondrial activity (MTT) was significantly lower at all concentrations in both methods, except at 1:1000 in method 1. However, the lysosome viability (NR) results revealed cytotoxicity only in the 1:1 sample of method 1. Both culture preparation methods were efficient and sensitive to the MTT assay, but method 2 seemed to be more adequate for the NR assay. The Rolante River has cytotoxic contaminants to Hep-2 cells, which may be one of the explanations for the poor water quality of the Sinos River basin.Keywords: cytotoxicity, Sinos River basin, samples preparation. Ensaios de citotoxicidade como ferramentas para avaliar a qualidade da água da bacia do Rio do Sinos ResumoOs ensaios de citotoxicidade utilizando culturas de células constituem uma alternativa para avaliar a toxicidade biológica de águas de superfície e podem auxiliar no controle da qualidade da água. Este estudo comparou dois métodos de preparação dos meios de cultura com amostras de água coletadas no rio Rolante, um importante afluente do Rio dos Sinos, para a exposição de células Hep-2. A toxicidade foi avaliada usando os ensaios do MTT e do vermelho neutro (VN). Dois métodos foram utilizados para preparar os meios de cultura. No método 1, a amostra foi diluída a 1:1, 1:10, 1:100, 1:1000 e 1:10.000 (v/v, amostra/ meio de cultivo) em um meio de cultura padrão; no método 2, as amostras de água foram utilizados como solventes para o meio de cultura, o qual foi preparado em concentração de 100% e nas diluições de 80, 60, 40 e 20%. Culturas semi-confluentes foram então expostas aos meios teste durante 24 horas, e a citotoxicidade foi determinada imediatamente usando os ensaios MTT e VN. A atividade mitocondrial (MTT) foi significativamente menor em todas as concentrações em ambos os métodos, exceto na diluição 1:1000 do método 1. No entanto, os resultados de viabilidade lisossomal (VN) revelaram citotoxicidade apenas no na diluição 1:1 do método 1. Ambos os métodos de preparação do meio cultura foram eficientes e sensíveis para o ensaio do MTT, mas o método 2 foi mais adequado para o ensaio do VN. O rio Rolante possui contaminantes citotóxicos para as células Hep-2, o que pode ser uma...

Photocatalytic ozonation of urban wastewater and surface water using immobilized TiO2 with LEDs: Micropollutants, antibiotic resistance genes and estrogenic activity

“…The first two cell lines are human-derived, while L929 fibroblasts were originated in mouse, accounting for their different susceptibility. Furthermore, as reported before (Trintinaglia et al, 2015), the exposure to water testing sample mixed with culture media can originate different results, depending upon the final growth media composition.…”

“…Furthermore, cytotoxicity and cellular viability assays using cell models for digestive epithelium (Caco-2), bloodbrain barrier (hCMEC/D3), and skin (L929) showed no significant difference concerning cell viability (Table 3, LDH assay). MTT assay is a suitable tool for assessment of cytotoxic effects from wastewater, surface water and drinking water samples as reported before (Trintinaglia et al, 2015;Z � egura et al, 2009). Slight cytotoxicity was observed for Caco-2 and hCMEC/D3 cell lines after treatment, but not for L929 fibroblasts (Table 3, MTT assay).…”

Original Article. Toxicity of Glandularia selloi (Spreng.) Tronc. leave extract by MTT and neutral red assays: influence of the test medium procedure

“…In order to evaluate cytotoxicity using the mitochondrial activity parameter, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used as described by Mosmann ( 1983 ). After exposure for 24 hours, the assay was performed according to Trintinaglia et al ( 2015 ). Briefly, 20 μL of MTT 1.67 mg/mL (Sigma Aldrich) was added to the well and the plates were incubated for two hours.…”

“…The neutral red (NR) incorporation method, described by Borenfreund and Puerner ( 1985 ), was used to evaluate cytotoxicity through lysosome viability. After exposure for 24 hours, the assay was performed according to Trintinaglia et al ( 2015 ). Briefly, the culture medium was removed and 200 μL of DMEM without serum and containing 50 μg/mL of NR (Sigma Aldrich) was added to each well.…”

A Pilot Study Combining Ultrafiltration with Ozonation for the Treatment of Secondary Urban Wastewater: Organic Micropollutants, Microbial Load and Biological Effects

“…Cytotoxic and cell viability effects of wastewater collected before (WW) and after treatment with O 3 + UF (TWW 0 ) were evaluated for skin (L929) and digestive epithelium (Caco-2) cell models by performing complementary MTT and LDH assays ( Table 3). Considering that cell viability upon exposure to water samples depends on the final composition of the growth medium [62], test samples were diluted 5 and 10 times in culture medium before incubation with cell layers. Similar cytotoxicity (MTT) and cell viability (LDH) values were obtained for both dilution levels (Table 3).…”